Defining the Catalytic Mechanism of the HDV Ribozyme. The hepatitis delta virus (HDV) is a human pathogen that causes severe acute and chronic liver disease. The viral genome contains the sequence for a ribozyme that is essential for replication. Although our understanding of ribozyme catalysis has advanced enormously in the two and half decades since their discovery, their mechanisms of catalysis remain largely undefined. Although crystal structures for many of these ribozymes have been solved, significant discrepancies between the structural data and functional experiments remain with many ribozyme systems including the HDV ribozyme. Additionally transition state structure for most ribozymes remains largely undetermined. In this proposal we will employ powerful chemical mutagenesis approaches together with atomic mutation cycles to reveal atomic connections in the molecule that are critical for function, including the connections between the ribozyme, the catalytic groups, and the reaction transition state. Concurrently, we will apply newly developed state of the art techniques in kinetic isotope effect (KIE) analysis (in collaboration with Michael Harris at Case Western Reserve University) to characterize the reaction pathway and obtain a measure of the bonding changes that occur during catalysis. We will then combine atomic mutagenesis with KIE measurements to determine how functional interactions in the ribozyme influence bonding in the transition state. This combination of approaches is unprecedented in RNA enzymology and promises to yield penetrating new insights into the catalytic mechanism of this RNA. Overall this work will establish an experimental and conceptual paradigm for studying other RNA enzymes and nucleotidyl transferases and the implications for biological catalysis will be far-reaching.

Public Health Relevance

HDV is a human pathogen causing severe acute and chronic liver disease in humans. The viral genome contains a ribozyme (HDV ribozyme) required for viral replication, and the human genome contains a similar sequence of unknown function. Understanding the ribozyme's mechanism of action promises to shed light on fundamental aspects of RNA catalysis and reveal critical features of viral pathogenicity, which may lead to new treatments for hepatitis.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI081987-05
Application #
8663828
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Koshy, Rajen
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Chicago
State
IL
Country
United States
Zip Code
60637
Koo, Selene C; Lu, Jun; Li, Nan-Sheng et al. (2015) Transition State Features in the Hepatitis Delta Virus Ribozyme Reaction Revealed by Atomic Perturbations. J Am Chem Soc 137:8973-82
Weissman, Benjamin P; Li, Nan-Sheng; York, Darrin et al. (2015) Heavy atom labeled nucleotides for measurement of kinetic isotope effects. Biochim Biophys Acta 1854:1737-45
Kellerman, Daniel L; Simmons, Kandice S; Pedraza, Mayra et al. (2015) Determination of hepatitis delta virus ribozyme N(-1) nucleobase and functional group specificity using internal competition kinetics. Anal Biochem 483:12-20
Lu, Jun; Koo, Selene C; Li, Nan-Sheng et al. (2015) Synthesis of 2'-O-photocaged ribonucleoside phosphoramidites. Nucleosides Nucleotides Nucleic Acids 34:114-29
Suslov, Nikolai B; DasGupta, Saurja; Huang, Hao et al. (2015) Crystal structure of the Varkud satellite ribozyme. Nat Chem Biol 11:840-6
Harris, Michael E; Piccirilli, Joseph A; York, Darrin M (2015) Integration of kinetic isotope effect analyses to elucidate ribonuclease mechanism. Biochim Biophys Acta 1854:1801-8
Chen, Haoyuan; Piccirilli, Joseph A; Harris, Michael E et al. (2015) Effect of Zn2+ binding and enzyme active site on the transition state for RNA 2'-O-transphosphorylation interpreted through kinetic isotope effects. Biochim Biophys Acta 1854:1795-800
Huang, Hao; Suslov, Nikolai B; Li, Nan-Sheng et al. (2014) A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore. Nat Chem Biol 10:686-91
Kellerman, Daniel L; York, Darrin M; Piccirilli, Joseph A et al. (2014) Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2'-O-transphosphorylation. Curr Opin Chem Biol 21:96-102
Gu, Hong; Zhang, Shuming; Wong, Kin-Yiu et al. (2013) Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2'-O-transphosphorylation. Proc Natl Acad Sci U S A 110:13002-7

Showing the most recent 10 out of 23 publications